7th WORKING GROUP MEETING “Integrated Protection of Olive Crops”

A. Xylella fastidiosa1. The threat posed by Xylella fastidiosa to olive trees in the Mediterranean region: thefirst outbreak in Apulia, ItalyDr. Anna Maria D’Onghia from Istituto Agronomico Mediterraneo di Bari, Valenzano (BA),Italy (CIHEAM)Analysed thoroughly the current situation of Xylella fastidiosa in Italy, (a known pathogen inAmericas but not previously known in Europe) and stressed that the Mediterranean Oliveindustry is seriously threatened by X. fastidiosa subspecies pauca strain “Codiro”. Nearly twoyears ago the pathogen was reported for the first time on olive trees in Apulia, in associationwith the olive quick decline syndrome (OQDS).Strain “Codiro”seems to be the main responsible agent, even though Phaeoacremonium andPhaemoniella, and Zeuzera pyrina could be also isolated or found.She underlined the possible association of organic olive culture of almost abandonedunfertilised, neither irrigated nor sprayed trees on the outbreak of the disease. As for thehost range the study is still in progress, but apparently the Codiro strain does not infectgrapevine and citrus spp. The assessed vector of the Codiro strain is Philaenus spumarius, apolyphagous Auchenorrhyncha insect widely distributed in the Euro-Mediterranean region

CONCLUDING REMARKS: PHYTOPATHOLOGICAL ISSUESprepared by Professor ERIS TJAMOS

Agricultural University of Athens

• There is no record of successful eradication of X. fastidiosa once established outdoors due to its broad range of plant hosts and vectors.

• Control strategies are mainly based on the prevention of introduction of the pathogen from areas where it is present and on the containment of the outbreak. She mentioned that no nursery material is allowed to me moved out of Puglia

• She noticed that areas where the cultivation does not show yet the symptoms of the disease have been properly cultivated iirigated fertilised and spraed with chemicals.

• Large-scale monitoring has been carried out in an attempt to contain the rapid spread of the pathogen and the correlated disease, which is destroying century-old olive trees.

• Efficient and innovative detection tools including monitoring methods have been developed (i.e. X. fastidiosa-detection on 'spy insects' for assessing the presence of the pathogen before symptom development, etc.). Based on these tools, an urgent pathogen surveillance programme should be envisaged in the Mediterranean countries to immediately monitor the presence of OQDS.

• Neighbor countries should be in alert•

2. Evaluation of "Insect Spy” approach for monitoring Xylella fastidiosa in symptomless olive orchards in the Salento Peninsula (Southern Italy)

Issam Eddine Ben Moussa1,2, Franco Valentini2, Donato Lorusso2, Valerio Mazzoni3,

Michele Digiaro2, Leonardo Varvaro1, Anna Maria D’Onghia2 by Viterbo, and Valenzano CIHEAM

• They developed an effective approach for monitoring this bacterium inthe symptomless areas surrounding the infection sites (buffer zone).

• Since the bacterium is vector borne, an evaluation of the possibility toutilize the Xf-positive insects found in the buffer zones (so called “InsectSpy”) as indicators of the presence of the bacterium in apparentlyuncontaminated areas, was carried out. The bacterium was successfullydetected by PCR in 3 out of 6 species of Auchenorrhyncha captured in thebuffer zone i.e. Phileanus spumarius, Neophileanus campestris andEuscelis lineolatus.

• Infected adults of these species were detected throughout the bufferzone at a distance of 1 Km far from the infection site.

• Therefore, adults of these three species have an important role as“insects spy” to early reveal the presence of the pathogen in apparentlyfree areas before symptoms become visible on the plants

3. Risk assessment of Xylella fastidiosa for olive growing area of CroatiaMario Bjeliš, Dario Ivić

Croatian Centre for Agriculture, Food and Rural Affairs, Institute for Plant Protection, Zvonimirova 14 a, 21210 Solin, Croatia

• Recent outbreak of the harmful bacterium Xylella fastidiosa strain CoDiROrepresents a potential risk for Croatian olive growing sector.

• The diversity of susceptible hosts in the Croatian sea coast and the fact thatpopulation of the coastal region is traditionally engaged in agriculture andtourism, facilitates the uncontrolled transmission Furthermore five potentialvector species [Philaenus spumarius L., Phylaenus signatus L., Lepyroniacoleoptrata L. and Aphrophora alni F. (Hemiptera: Aphrophoridae) and Cicadellaviridis L. have been described in olive growing areas of the under study area.Results of the 2014 surveys in Croatia, after extensive visual inspections andlaboratory tests, indicated that Xylella fastidiosa strain CoDiRO is not present inCroatia.

• Presence of potential and confirmed vectors of the pathogen, abundance andhigh biodiversity of host plants, favorable climate and high movement of plantmaterial are important elements that should be taken into account for the riskassessment of the spread of pathogen in case of his undesirable introduction.

•

4. Quick decline syndrome Xylella fastidiosa and anthracnose: emerging and reemerging diseases of olive posing new challenges for an integrated control approach

Franco Nigro, Ilaria Antelmi, Antonio Ippolito from Università degli Studi di Bari – Aldo Moro, via Amendola 165/A, 70126 Bari, Italy

• Xylella fastidiosa vectored by Cicadellidae insects,affects young and aged trees (100 years or older)growing in the Salento peninsula.

• Fungal species of the genus Phaeoacremoniunand several other isolates similar to genusPhaeomoniella were obtained from thediscolored wood.

• The role of these fungi, alone or in combination,in determining the symptoms observed on olivetrees is currently under investigation.

B. Colletotrichum acutatum1. Serious anthracnose outbreak starting from inflorescence infection of olive

trees in west Greece Colletotrichum accutatum Eleftherios C. Tjamos1, Maria C. Iliadi2, Dimitrios I. Tsitsigiannis2, Polymnia P.

Antoniou21Hellenic Plant Clinic Center, Greece2 Laboratory of Plant Pathology, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece

• Anthracnose is very common and particularly destructive in olive groves of westGreece, along the west coastal area and the Ionian Islands due to prolonged wetperiods because of heavy rains. In Aetolia region last year almost the 25% ofthe expected production of Kalamata was destroyed. Orchard surveys lastJanuary showed mummified fruits on the trees. Similar symptoms and signswere easily observed in olive fruits already fallen on the soil.

• Surveys in Zakynthos island on March 26, 2015 revealed that certain olivegroves of the variety Koroneiki at the onset of blooming developed a dark red tobrown discoloration demonstrated formation of acervuli (conidiosomata). ofthe colonies and the dimension and form of the conidia that we deal with thespecies Colletotrichum accutatum.

• Similar symptoms were also observed in in April 18 2015 in Kalamata county inKoroneiki variety. Molecular identification of the pathogen is under examination.

• Our research group is currently organizing field and lab experiments to furtherstudy the problem and find out the best possible measures and methods ofdisease management.

2. Biological control of olive anthracnoseFranco Nigro1, Ilaria Antelmi1, Rossella Labarile1, Valentina Sion1, Isabella Pentimone2

1Dipartimento di Scienze del Suolo, Bari, Italy2Istituto per la Protezione Sostenibile delle Piante (IPSP), Consiglio Nazionale delle Ricerche,

Via Amendola 122/D, 70126 Bari, Italy

• The biocontrol activity of the commercial formulate Serenade®,based on Bacillus subtilis, and several endophytic isolates of theyeast-like fungus Aureobasidium pullulans were evaluated infield trials.

• Bacillus subtilis applied at the pre-blooming stage was effective as the chemical fungicides.

• Moreover, some endophytic A. pullulans strains provided high protection levels against Colletotrichum spp. when applied at the veraison stage.

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3. Endophytic fungi of olive tree and its potential application in protection against Colletotrichum acutatum

Fátima Martins, José Alberto Pereira, Albino Bento, Paula BaptistaCIMO / School of Agriculture, Polytechnic Institute of Bragança, Campus de Santa Apolónia,

5300-253 Bragança, Portugal

The diversity of fungal endophytes from roots, leavesand twigs of healthy trees from three olive treecultivars with different susceptibilities to oliveanthracnose were assessed to select strains with thegreatest antagonistic effect against Colletotrichumacutatum. (identifiy them by rDNA sequencing)

They suggest exploring them as biocontrol agentsand use the for elucidating the mechanism ofenhanced disease resistance in olive tree cultivars.

C. Spilocaea oleagina1. Relationship between phyllosphere fungal communities and tolerance of olive tree

cultivars to olive leaf spot disease Spilocaea oleagineaTeresa Gomes1,2, José Alberto Pereira1, Teresa Lino-Neto2, Paula Baptista1

1Mountain Research Centre (CIMO) / Polytechnic Institute of Bragança, Agrarian School, Campus Santa Apolonia, 5300-253 Bragança, Portugal 2Biosystems & Integrative Sciences Institute (BioISI), Plant Functional Biology Center (CBFP), University of Minho, Campus de Gualtar, 4710-057

Braga, Portugal

Spilocaea oleagina is the causal agent of olive leaf spot (OLS) disease on olive trees, A correlation between data regarding fungal community and disease incidence displayed by both cultivars was performed by using multivariate analyses.

Their results indicate that some fungal species, either epiphytes or endophytes, may have a direct effect on olive tree tolerance to Spilocaea oleaginea .

2. Diversity of epi- and endophytic fungi on asymptomatic and symptomatic olive tree phyllosphere

Teresa Gomes1,2, José Alberto Pereira1, Teresa Lino-Neto2, Paula Baptista1 Spilocaea oleagina

1Mountain Research Centre (CIMO) / Polytechnic Institute of Bragança, Agrarian School, Campus Santa Apolónia, 5300-253 Bragança, Portugal

2Biosystems & Integrative Sciences Institute (BioISI), Plant Functional Biology Center (CBFP), University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal

• Their work aimed at comparing the endo- and epiphyticfungal community inhabiting asymptomatic and symptomaticleaves of olive trees (Olea europaea L.) that showed typicalsymptoms of Spilocaea oleagina

• The plant material was collected from seven olive trees, in agrove located in Mirandela (Northeast Portugal), being usedto isolate endo- and epiphytic fungi.

• The presence of Spilocaea on leaves clearly affects fungalcommunity composition, which in turn may have implicationson olive tree protection against this disease. It is expect toidentify predictable microbial species that could explain thereduction of OLS disease infection in asymptomatic leaves.

D. Verticillium dahliae1. Verticillium wilt of olive - Current status and Management

Leah Tsror (Lahkim) Agricultural Research Organization, Gilat Research Center, Department of Plant Pathology,

85250, Israel

• She stressed that we need an integrated diseasemanagement (IDM) to reduce disease impact, by usingpreventive (V. dahliae -free soil and planting material),pre-planting (resistant cultivars, soil disinfestation,sanitation) and post-planting (cultural practices, soilsolarization, organic amendments, green manure,biological control agents) measures.

• Control of Verticillium wilt on olive is an ongoingchallenge which requires further research forunderstanding the pathogen-tree-environment

2. Genomic analysis of two Pseudomonas spp. strains displaying endophytic lifestyle in olive roots and effective biological control of Verticillium dahliae

Pedro M. Martínez-García1, 3, David Ruano-Rosa2, Elisabetta Schiliro`2, Carmen Gómez-Lama Cabanás2, Cayo Ramos1, Pablo Rodríguez-Palenzuela3, Jesús Mercado-Blanco2

1Instituto de Hortofruticultura Subtropical y Mediterránea - Universidad de Málaga - Agencia Estatal Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Málaga, Spain

2Departmento de Protección de Cultivos, Instituto Agricultura Sostenible (CSIC), Córdoba, Spain3Centro de Biotecnología y Genómica de Plantas (UPM- INIA), Pozuelo de Alarcón, Madrid, Spain

•

• Pseudomonas fluorescens PICF7 and Pseudomonas putida PICP2 are endophytesand effective biocontrol strains against Verticillium dahliae of olive.

• They sequenced and annotated the complete genomes of strains PICF7. Acomparative bioinformatics analysis was carried out aiming to identify genespotentially involved in

• (i) biological control,• (ii) plant-bacteria interaction,• and (iii) endophytism.• The analysis has revealed that, the genome of strain PICF7 harbours the genetic

information to encode two type VI (T6SS) and one Type III (T3SS) secretionsystems, the T3SS effectors avrE1 and hopB1, the siderophores pyoverdine,pyochelin and the hemophore HasAp, diverse adhesion factors, a battery of cell-degrading enzymes (protease, cellulase and lipase), as well as detoxifying systemssuch as CopA, KatB, Dps and Cb), and AGL2011-30343-CO2-01 from the MINECO(Spain), all co-funded by ERDF from EU.

3. Influence of irrigation frequency on the onset and development of Verticillium wilt of oliveMario Pérez-Rodríguez1, Nicolás Serrano2, Ignacio J. Lorite2, Octavio Arquero2, Francisco

Orgaz3, Francisco Javier López-Escudero1

1Dpto. Agronomía, Universidad de Córdoba. Campus Rabanales, Edificio C4, 14071 Córdoba, Spain2IFAPA, Centro Alameda del Obispo, 14080 Córdoba, Spain

3Instituto de Agricultura Sostenible , CSIC Alameda del Obispo, 14080 Córdoba, Spain

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• The Spanish Research Group AGR-216 from the University ofCordoba has conducted 4-year experiments for studying theeffect of irrigation on development of Verticillium wilt of olive.

• During the evaluation period results clearly demonstrated thatirrigation treatments encouraged Verticillium wilt developmentwhen compared to the deficit irrigation.

• They concluded that, up to 52 weeks after planting, values ofthe area under disease progress curve (AUDPC) weresignificantly higher in daily irrigated trees (15.8%) than in oliveplants of the other two treatments (5.0%).

4. VERTICILLIUM DAHLIAECurrent problems in managing Verticillium wilt of olives in Greece and the prospective of non

chemical and biological control of the disease in olive orchardsPolymnia P. Antoniou, Sotirios E. Tjamos, Dimitrios I. Tsitsigiannis, Eleftherios C. Tjamos

Laboratory of Plant Pathology, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece

• Biological control of Verticillium wilt was attempted in heavily infested oliveorchards of Kalamata variety by drenching the soil around the trees withbacterial suspensions of Paenibacillus alvei strain K-165, effective againstVerticillium wilt of potatoes in the field. It was shown that soil application ofthe biocontrol agent applied at 4 month intervals mitigated significantlysymptoms of the disease. New infestation of a year after was attributed tothe incorporation of leaves from diseased trees during the next years afterthe treatments, underlying the importance of the inoculum level inovercoming the effectiveness of biological control agents.

• The application of soil solarization in already established olive orchardsproved that heavy inoculum coming mainly from infested leaves aredrastically reduced so solarization is a promising and effective method todecrease the soil inoculum level of the pathogen in young olive orchards.

5. Screening of organic amendments, plant extracts and microorganisms for the control of Verticillium wilt in olive trees

Ángela Varo, Carmen Raya, Luis F. Roca, Antonio Mulero, Mustafa Adem, Francisco Javier López-Escudero, Antonio Trapero

Dept. Agronomía, Universidad de Córdoba. Campus Rabanales, Edificio C4, 14071 Córdoba, Spain

• Different organic amendments plant extracts and microorganisms(various fungi, bacteria and their extracts) for the control of V. dahliaewere selected.

• The selection was conducted in four stages:• i) in vitro, by the effect on the mycelial growth and spore germination of

the pathogen;• ii) in naturally-infested soil, by the effect on the reduction of

microsclerotia of the pathogen;• iii) in planta, by the effect on the infection of olive plants under

controlled conditions; and iv), in field, by the effect on Verticillium wiltof olive trees grown in a highly-infested soil.

• The assessment of 220 treatments under controlled conditions resultedin a selection of 20 candidates to evaluate in field conditions. Among thecandidates evaluated in the field, are non-pathogenic strains of Fusariumoxysporum, pomaces of grape and olives, and mixtures of bacteria andyeasts.

6. Diversity of bacteria endophytes in olive treeDiogo Mina, José Alberto Pereira, Albino Bento, Paula Baptista

Mountain Research Centre (CIMO), School of Agriculture, Polytechnic Institute of BragançaCampus de Santa Apolónia, 5300-253 Bragança, Portugal

• Their work intends to assess the diversity of culturable bacterial endophytes in O. europaea and create a reservoir of strains with potential beneficial applications on olive tree protection against Verticillium wilt.

• Bacterial endophytes were isolated from roots, leaves and twigs of 35 olive trees cv. Picual, located in Granada (Spain). Pure bacteria cultures were identified morphological- and molecularly through sequencing of V1 to V4 regions from 16S rDNA, and further maintained in culture (PCA) and cryopreserved (-80ºC in aqueous glycerol solution). A total of 35 taxa belonging to 14 genera were identified. The bacterial endophytes most frequently observed were Alcaligenesfaecalis and Pseudomonas aeruginosa, found in 9% of roots and 80% twigs, and in 8% of roots, respectively. Root explants were the most colonized (96%), followed by twigs (3%) and leaves (1%).

• Correspondence analysis of the endophyte assemblages showed that the endophytes exhibited organ specificity.

• Thirty three species were consistently associated with root tissues, while one species was found predominantly in leafs.

• Many bacterial species are found to be restricted to root tissues, and knowledge of their interactions with V. dahliae might be useful in the control of this disease.

7. VERTICILLIUM DAHLIAEPruning residue management associated pathogens in olive

Emmanouil Markakis, George Koubouris, Nektarios Kavroulakis, George Psarras, Chryssa Sergentani, Argyro Kalaitzaki

ELGO DEMETER, NAGREF, Institute for Olive Tree, Subtropical Crops and Viticulture, Agrokipio, 73100 Chania, Greece

• They investigated whether recycling of raw or compostedpruning residues in soil could potentially contribute to fungalpathogens dispersal, such as Verticillium dahliae and Fomitiporiamediterranea, within and between olive orchards. (DNAextraction and polymerase chain reaction-PCR assay) wereemployed to detect the abovementioned pathogens in healthyand diseased plant tissues as well as in raw or composted plantmaterials. Both fungal species were detected in diseased tissuesbut not in composted materials.

• The outcome of these analyses indicates the low risk ofpathogen dispersion in the fields following good agriculturalpractices and regular orchard monitoring.